SUMMARY1. Single ventricular cells were enzymatically isolated from adult guinea-pig hearts (Isenberg & Kiockner, 1982). The patch-clamp technique (Hami1lWMarty, Neher, Sakmann & Sigworth, 1981) was used to examine the conductance properties of an inward-rectifying K+ channel present in their sarcolemmal membrane.2. When the K+ concentration on the extracellular side of the patch was between 10-8 and 300 mm, inward current steps were observed at potentials more negative than the K+ equilibrium potential (EK). At more positive potentials no current steps were detectable, demonstrating the strong rectification of the channel.3. The zero-current potential extrapolated from the voltage dependence of the inward currents depends on the external K+ concentration [K+] Assuming a Michaelis-Menten scheme for binding of permeating K+ to the channel, an apparent binding constant of 210 mm is calculated for a membrane potential of -100 mV. For this potential the current at saturating [K+]o is estimated as 6-5 pA.6. The rectification of the single-channel conductance at membrane potentials positive to EK occurs within 1-5 ms of stepping the membrane potential from a potential of high conductance to one of low conductance. 7. In addition to the main conductance state, the channel can adopt several substates of conductance. The main state could be the result of the simultaneous opening of four conducting subunits, each of which has a conductance of about 7 pS in 145 mM-external K+. B. SAKMANN AND 0. TRUBE 8. The density of the inward-rectifying K+ channels in the ventricular sarcolemma is 0-10 channel/10 pm2 of surface membrane; the average of twenty-eight patches was 1 channel/1i8,Um2.9. It is concluded that the inward-rectifying K+ channels mediate the resting K+ conductance of ventricular heart muscle and the current termed IKi in conventional voltage-clamp experiments.

No abstract

Venom-derived peptide toxins can modify the gating characteristics of excitatory channels in neurons. How they bind and interfere with the fl ow of ions without directly blocking the ion permeation pathway remains elusive. Here we report the crystal structure of the trimeric chicken Acid-sensing ion channel 1 in complex with the highly selective gating modifi er Psalmotoxin 1 at 3.0 Å resolution. The structure reveals the molecular interactions of three toxin molecules binding at the proton-sensitive acidic pockets of Acid-sensing ion channel 1 and electron density consistent with a cation trapped in the central vestibule above the ion pathway. A hydrophobic patch and a basic cluster are the key structural elements of Psalmotoxin 1 binding, locking two separate regulatory regions in their relative, desensitized-like arrangement. Our results provide a general concept for gating modifi er toxin binding suggesting that both surface motifs are required to modify the gating characteristics of an ion channel.

SUMMARY1. Inward currents through single K+ channels in isolated ventricular heart cells of the guinea-pig were recorded using the patch-clamp techhi-que(Hamill, Marty, Neher, Sakmann & Sigworth, 1981). The voltage-dependent gating properties of the channels were examined in the potential range between 0 and -120 mV with 145 mM-KCl on the extracellular side of the membrane patch, i.e. with approximately symmetrical transmembrane K+ concentrations.2. When voltage pulses from 0 mV to negative test potentials were applied to patches containing several channels, more channels were open at the beginning of the pulses than in the steady state. Averages of many current responses showed inactivation of the mean current in response to the hyperpolarizing voltage pulses. The inactivation was stronger and faster at larger hyperpolarization.3. The lifetimes of the open and closed states of the channel and the probability of the open state p were estimated from records of the elementary currents at various constant potentials. As indicated by the inactivation of the averaged currents, the value of p was smaller at more negative potentials, approximately 0 15 at -50 mV and 0-02 at -110 mV. This caused a negative slope in the current-voltage relation of the time-averaged current at potentials more negative than -50 mV.4. The channel openings were grouped in complex bursts. At least three exponentials were needed to fit the frequency histogram of the lifetimes of all closed states (time constants at -50 mV: 1-1 ms, 16 ms and 3-2 s). The lifetimes of the individual openings were exponentially distributed (time constant: 70 ms).5. The kinetics of the channel were interpreted by two different models involving three states of a channel (closed-closed-open or closed-open-closed). The rate constants and their voltage dependence were estimated for both models. Both models describe the data equally well; the reason for this ambiguity is discussed.6. The channels are blocked by Cs+ or Ba2+. Cs+ (0.1 mM) caused frequent and short interruptions of the individual channel openings. Ba2+ (0-5 mM) also shortened the openings and in addition decreased the number of openings per burst.

Inward rectifying potassium single-channel currents were studied in the membrane of guinea pig cardiac myocytes. In isolated inside-out patches two different channels were observed: a channel of 25 pS conductance ([K+]o = 147 mM, T = 21 degrees C), if the solution at the cytoplasmic face of the patch contained 4 mM ATP and a channel of 80 pS conductance without ATP. The 25-pS-channel was also regularly seen in cell-attached patches (Sakmann and Trube 1984a,b), but the 80-pS-channel appeared only after inhibiting cellular metabolism by DNP. The percentage of time which the 25-pS-channel spent in the open state was 3.3 times larger in isolated patches compared to cell-attached patches. However, both types of single channel currents disappeared several minutes after the isolation of the patches. In contrast to the 25-pS-channel, the 80-pS-channel, which is activated by the lack of ATP, carried measurable outward currents saturating at 1.5 pA (inward rectification). It is suggested that the 80-pS-channel mediates part of the increase in potassium current during metabolic inhibition. The openings of this channel appeared in bursts. The mean open time was 1.6 ms and the mean duration of the gaps within bursts 0.33 ms at -80 mV.